Toll-like receptors (TLRs) have evolved to recognize conserved features of microbial pathogens. Several TLR family members recognize nucleic acids as signatures of viral infection. For example, TLR9 recognizes unmethylated CpG DNA motifs present in many DNA viruses. While ensuring the detection of pathogens, this specificity for nucleic acids also exposes the host to potential autoimmunity due to inappropriate recognition of self nucleic acid. Thus, mechanisms must exist that prevent self- recognition while still allowing detection of foreign nucleic acid. In this context, it is striking that all the TLRs involved in nucleic acid sensing are retained intracellulariy, while other TLRs are expressed at the cell surface. We hypothesize that the intracellular compartmentalization of TLR9 prevents the recognition of self-DNA. The overall goal of this proposal is to define the mechanisms that establish and maintain TLR9 trafficking and localization. In Aim 1, we will define the motif/s within TLR9 that govern trafficking to the endolysosome using truncations of TLR9 as well as full-length TLR9 with selected mutations. In Aim 2, we will identify cellular factors that control endolysosomal localization of TLR9 using a candidate gene approach as well as an siRNA screen. The completion of this proposal will result in a better understanding of TLR9 cell biology and will have implications for self/non-self discrimination by this receptor.